Mitochondria and their Role in Apoptosis
Mitochondria are central to basic life functions, particularly the generation of cellular energy, and as such are the sites of key components of the biosynthetic pathways. Mitochondria are also potent integrators and coordinators of programmed cell death or apoptosis. The mitochondrion represents a discrete sub-cellular organelle that is comprised of about 1000 different protein species. One of these mitochondrial proteins controlling processes associated with both cell life and death is the voltage-dependent anion channel (VDAC).
The role of mitochondria in regulating cell death has been revealed and it is well accepted that mitochondria play a major role in regulating apoptosis (Kroemer et al. (1997). Immunol. Today 18, 44-51). Following an apoptotic stimulus, various proteins that normally reside in the intermembrane space of mitochondria, including cytochrome c and apoptosis-inducing factors, are released to the cytosol and initiate the activation of procaspases, the protease mediators of cell death. It remains unclear, however, how these apoptosis initiators cross the outer mitochondrial membrane (OMM) and are released to the cytosol. While some models predict that release occurs as a result of the swelling of the mitochondrial matrix with subsequent rupture of the OMM, other models predict the formation of a pore large enough to allow the passage of cytochrome c and other proteins into the cytosol, without compromising OMM integrity. The finding that cytochrome c can leak from intact mitochondria (see, for example, Doran and Halestrap (2000). Biochem J. 348 (Pt 2), 343-350) supports those models predicting the specific permeability of the OMM. VDAC, previously suggested to participate in the release of cytochrome c, is considered as a promising candidate for such an OMM pore-forming protein. Some of the inventors of the present invention and co-workers have shown that VDAC functions in the release of apoptotic-mediated proteins from the mitochondria and to interact with different apoptosis-regulators (Shoshan-Barmatz et al. (2006) Curr. Pharm. Design, 12, 2249-2270; Shoshan-Barmatz and Israelson (2005) J. Membr. Biol. 204, 57-66).
VDAC, the major OMM transporter, also plays an important role as a controlled passage of adenine nucleotides, Ca+ and other metabolites into and out of mitochondria, thereby, may also function as a key protein in the energy production by mitochondria (Colombini (2004). Mol Cell Biochem 256-257 (1-2), 107-115).
VDAC: Structure-Function
Mammalian VDAC, also known as mitochondrial porin, is a 31 kDa protein with a large pore diameter of about 3.0 nm. VDAC has been purified and characterized by reconstitution into a planar lipid bilayer (PLB), and its conductance and ion selectivity were found to be voltage dependent (Shoshan-Barmatz and Gincel (2003) Cell Biochemistry and Biophysics 39, 279-292).
The molecular nature of VDAC gating mechanism has yet to be resolved. Computer modeling of the VDAC's primary amino acid sequences led to the development of models showing the transmembrane organization, consisting of a single amphipathic N-terminal α-helix and 13 or 16 transmembrane β-strands (Colombini (2004), supra; De Pinto et al. (2003) Ital. J. Biochem. 52, 17-24). These β-strands are connected by several peptide loops of different sizes on both sides of the membrane that serve as potential protein interacting sites. It is widely accepted that monomeric VDAC serves as the functional channel in Saccharomyces cerevisiae (Peng et al. (1992) Bioenerg Biomembr 24, 27-31). However, evidence, consistent with oligomerization of rat liver or Neurospora crassa purified VDAC, suggests that VDAC exists as a dimer, and possibly a tetramer (Krause et al. (1986) Biochim Biophys Acta, 860: 690-698). Using rat liver, brain mitochondria or recombinant human VDAC, the existence of VDAC dimers to tetramers was also reported (Zalk et al. (2005) Biochemical J. 386, 73-83; Shoshan-Barmatz et al. (2006) Curr. Pharm. Design, 12, 2249-2270).
Three mammalian isoforms of VDAC are known, VDAC1, VDAC2, VDAC3, where VDAC1 is the major isoform expressed in mammalian cells.
U.S. Pat. No. 5,780,235 discloses two novel VDAC sequences, which were named HACH (human voltage-dependent anion channel), subsequently identified as VDAC2 and VDAC3. That patent provides genetically engineered expression vectors, host cells containing the vector, a method for producing HACH, a method for identifying pharmaceutical compositions inhibiting the expression and activity of HACH and the use of such compositions for the treatment of cancer and proliferative diseases.
International Application Publication No. WO 2006/095347 to some of the inventors of the present invention discloses VDAC1 molecules capable of modulating apoptosis in a cell, the molecule selected from: (i) an isolated VDAC1 polypeptide variant having at least one amino acid substitution in an amino acid residue residing in a VDAC1 cytosolic domain and (ii) an isolated VDAC1 peptide fragment, analog, chemical derivative and a salt thereof, wherein the peptide fragment, analog, chemical derivative or salt thereof is derived from a VDAC1 cytosolic domain or partial sequence thereof. The amino acid sequences disclosed therein were shown to cause a dramatic increase in apoptosis of human cancer cell lines and in chemo- and radio-resistant cancer cells. A VDAC1 N-terminal domain peptide was disclosed as useful to enhance apoptosis.
All VDAC membranal topology predictions so far performed have emphasized the presence of a segment of α-helix, with amphipathic features, at the N-terminus of the protein. According to the three proposed models, the VDAC1 N-terminus amphipathic α-helix is either exposed to the cytoplasm (De Pinto et al. (2003). Ital. J. Biochem. 52, 17-24), crosses the membrane (Colombini (2004), Mol Cell Biochem 256-257, 107-115) or lies on the membrane surface (Reymann et al. (1995). Biochemical and Molecular Medicine, 54, 75-87). Although the N-terminal α-helix has been proposed to form part of the lumen facing the wall of the open state of the channel and shows some motion during voltage gating, the limited hydrophobicity of the sequence suggests it unlikely that this segment of VDAC is permanently embedded in the membrane.
Certain N-terminal variants of VDAC have been disclosed in the art. VDAC1 with N-terminal extensions display voltage-induced partial closures, while variants with an N-terminal region shortened by more than 6-7 amino acid residues exhibit a destabilized open state In addition, scVDACD1-8, the bacterially expressed truncated yeast variant lacking the first eight amino acids, behaves atypically in bilayer experiments: rather than forming stable open channels with discrete transitions to a lower subconductance level, scVDACD1-8 induces channels that flicker rapidly (Koppel et al. (1998). J. Biol. Chem. 273, 13794-800).
The role of the N and C-termini in channel formation was studied in Neurospora crassa VDAC (Popp et al. (1996). J. Biol. Chem. 271, 13593-13599). Wild-type and mutant porins from Neurospora crassa were expressed as His-tag fusion products. Mutants lacking part of the N-terminus (DeltaN2-12porin, DeltaN3-20porin), part of the C-terminus (DeltaC269-283porin), or both (DeltaN2-12/DeltaC269-283porin) showed channel-forming activity. A VDAC mutant or peptide useful for the inhibition of apoptosis was neither taught nor suggested. The N-terminal α-helix was shown to interact specifically with cytochrome c (Stanley et al. (1995). J Biol Chem 270, 16694-16700). In addition, it has also been suggested that the mobility of the α-helix may modulate the accessibility of the pro- and anti-apoptosis proteins Bax and Bel-XL to their binding sites at the VDAC loop regions (Shi et al. (2003). Biochem Biophys Res Comm 305, 989-96). Thus, the accumulated evidence suggests that the N-terminal region of VDAC corresponds to a mobile component of the protein.
U.S. Patent Application Publication No. 20050234116 discloses a promoter of neurotrophin (nerve growth factor) production/secretion as a VDAC regulator, apoptosis suppressor or mitochondria function ameliorator, useful as an agent for the prophylaxis or treatment of Down's syndrome and the like.
U.S. Patent Application Publication No. 20060252822 provides a new class of compounds for the labeling and modulation of mitochondrial permeability transition pore (MPTP) in the sub μM range, wherein isoform 1 of VDAC (VDAC1) is identified as a MPTP component and as the molecular target of these compounds. That disclosure further provides methods for identifying an active agent that modulates the activity of the MPTP complex, specifically methods for identifying an active agent that modulates the activity of the MPTP complex by interacting with the VDAC1 component.
The Role of GXXXG Motifs in the Function of the N-Terminus of VDAC1
Association between monomers is mediated by helix-helix contacts involving specific amino acid residues. The GXXXG motif and “GXXXG-like” motifs (in which one or both glycine residues are substituted by other small amino acids) has been identified as one of the most frequently occurring transmembrane (TM) sequence motifs as a potential site for tight interaction between TM α-helices (Arselin et al. (2003). Eur J Biochem 270, 1875-1884). This motif is a potential site for tight interaction between α-helices as a result of a three amino acid residue separation between the glycine residues aligns them on one face of the helix, thus providing a flat platform for close binding of a partner α-helix (Polgar et al. (2004). Biochemistry 43, 9448-9456). The GXXXG motif has been linked with dimerization of proteins including glycophorin A, human carbonic anhydrase, yeast ATP synthase and more. In VDAC, the GXXXG motif is present in the N-terminus of the channel that forms an α-helix structure.
There remains an unmet need for therapeutic agents effective in inhibiting or decreasing apoptosis that may be useful for the treatment of variant diseases, including neurodegenerative, cardiac and ophthalmic diseases. The art neither teaches nor suggests an N-terminal VDAC variant polypeptide or peptides useful for promoting cell proliferation and or inhibiting apoptosis.